Combinatorial action of NF-Y and TALE at embryonic enhancers defines distinct gene expression programs during zygotic genome activation in zebrafish.

Animal embryogenesis is initiated by maternal factors, but zygotic genome activation (ZGA) shifts regulatory control to the embryo during blastula stages. ZGA is thought to be mediated by maternally provided transcription factors (TFs), but few such TFs have been identified in vertebrates. Here we report that NF-Y and TALE TFs bind zebrafish genomic elements associated with developmental control genes already at ZGA. In particular, co-regulation by NF-Y and TALE is associated with broadly acting genes involved in transcriptional control, while regulation by either NF-Y or TALE defines genes in specific developmental processes, such that NF-Y controls a cilia gene expression program while TALE controls expression of hox genes. We also demonstrate that NF-Y and TALE-occupied genomic elements function as enhancers during embryogenesis. We conclude that combinatorial use of NF-Y and TALE at developmental enhancers permits the establishment of distinct gene expression programs at zebrafish ZGA.

ETV4 transcription factor and MMP13 metalloprotease are interplaying actors of breast tumorigenesis.

BACKGROUND: The ETS transcription factor ETV4 is involved in the main steps of organogenesis and is also a significant mediator of tumorigenesis and metastasis, such as in breast cancer. Indeed, ETV4 is overexpressed in breast tumors and is associated with distant metastasis and poor prognosis. However, the cellular and molecular events regulated by this factor are still misunderstood. In mammary epithelial cells, ETV4 controls the expression of many genes, MMP13 among them. The aim of this study was to understand the function of MMP13 during ETV4-driven tumorigenesis. METHODS: Different constructs of the MMP13 gene promoter were used to study the direct regulation of MMP13 by ETV4. Moreover, cell proliferation, migration, invasion, anchorage-independent growth, and in vivo tumorigenicity were assayed using models of mammary epithelial and cancer cells in which the expression of MMP13 and/or ETV4 is modulated. Importantly, the expression of MMP13 and ETV4 messenger RNA was characterized in 456 breast cancer samples. RESULTS: Our results revealed that ETV4 promotes proliferation, migration, invasion, and anchorage-independent growth of the MMT mouse mammary tumorigenic cell line. By investigating molecular events downstream of ETV4, we found that MMP13, an extracellular metalloprotease, was an ETV4 target gene. By overexpressing or repressing MMP13, we showed that this metalloprotease contributes to proliferation, migration, and anchorage-independent clonogenicity. Furthermore, we demonstrated that MMP13 inhibition disturbs proliferation, migration, and invasion induced by ETV4 and participates to ETV4-induced tumor formation in immunodeficient mice. Finally, ETV4 and MMP13 co-overexpression is associated with poor prognosis in breast cancer. CONCLUSION: MMP13 potentiates the effects of the ETV4 oncogene during breast cancer genesis and progression.

Hox regulation of transcription: more complex(es).

Hox genes encode transcription factors with important roles during embryogenesis and tissue differentiation. Genetic analyses initially demonstrated that interfering with Hox genes has profound effects on the specification of cell identity, suggesting that Hox proteins regulate very specific sets of target genes. However, subsequent biochemical analyses revealed that Hox proteins bind DNA with relatively low affinity and specificity. Furthermore, it became clear that a given Hox protein could activate or repress transcription, depending on the context. A resolution to these paradoxes presented itself with the discovery that Hox proteins do not function in isolation, but interact with other factors in complexes. The first such "cofactors" were members of the Extradenticle/Pbx and Homothorax/Meis/Prep families. However, the list of Hox-interacting proteins has continued to grow, suggesting that Hox complexes contain many more components than initially thought. Additionally, the activities of the various components and the exact mechanisms whereby they modulate the activity of the complex remain puzzling. Here, we review the various proteins known to participate in Hox complexes and discuss their likely functions. We also consider that Hox complexes of different compositions may have different activities and discuss mechanisms whereby Hox complexes may be switched between active and inactive states.

A Gal4/UAS system for conditional transgene expression in rhombomere 4 of the zebrafish hindbrain.

BACKGROUND: The zebrafish is well established as a model organism for the study of vertebrate embryogenesis, but transgenic lines enabling restricted gene expression are still lacking for many tissues. RESULTS: We first generated the hoxb1a(ß-globin):eGFP(um8) line that expresses eGFP in hindbrain rhombomere 4 (r4), as well as in facial motor neurons migrating caudally from r4. Second, we generated the hoxb1a(ß-globin) Gal4VP16(um60) line to express the exogenous Gal4VP16 transcription factor in r4. Lastly, we prepared the UAS(ß-actin):hoxa3a(um61) line where the hoxa3a gene, which is normally expressed in r5 and r6, is under control of Gal4-regulated UAS elements. Crossing the hoxb1a(ß-globin):Gal4VP16(um60) line to the UAS(ß-actin):hoxa3a(um61) line drives robust hoxa3a expression in r4. We find that transgenic expression of hoxa3a in r4 does not affect hoxb1a expression, but has variable effects on migration of facial motorneurons and formation of Mauthner neurons. While cases of somatic transgene silencing have been reported in zebrafish, we have not observed such silencing to date, possibly because of our efforts to minimize repetitive sequences in the transgenic constructs. CONCLUSION: We have generated three transgenic lines that will be useful for future studies by permitting the labeling of r4-derived cells, as well as by enabling r4-specific expression of various transgenes.

TALE factors use two distinct functional modes to control an essential zebrafish gene expression program.

TALE factors are broadly expressed embryonically and known to function in complexes with transcription factors (TFs) like Hox proteins at gastrula/segmentation stages, but it is unclear if such generally expressed factors act by the same mechanism throughout embryogenesis. We identify a TALE-dependent gene regulatory network (GRN) required for anterior development and detect TALE occupancy associated with this GRN throughout embryogenesis. At blastula stages, we uncover a novel functional mode for TALE factors, where they occupy genomic DECA motifs with nearby NF-Y sites. We demonstrate that TALE and NF-Y form complexes and regulate chromatin state at genes of this GRN. At segmentation stages, GRN-associated TALE occupancy expands to include HEXA motifs near PBX:HOX sites. Hence, TALE factors control a key GRN, but utilize distinct DNA motifs and protein partners at different stages - a strategy that may also explain their oncogenic potential and may be employed by other broadly expressed TFs.

A tissue-specific, Gata6-driven transcriptional program instructs remodeling of the mature arterial tree.

Connection of the heart to the systemic circulation is a critical developmental event that requires selective preservation of embryonic vessels (aortic arches). However, why some aortic arches regress while others are incorporated into the mature aortic tree remains unclear. By microdissection and deep sequencing in mouse, we find that neural crest (NC) only differentiates into vascular smooth muscle cells (SMCs) around those aortic arches destined for survival and reorganization, and identify the transcription factor Gata6 as a crucial regulator of this process. Gata6 is expressed in SMCs and its target genes activation control SMC differentiation. Furthermore, Gata6 is sufficient to promote SMCs differentiation in vivo, and drive preservation of aortic arches that ought to regress. These findings identify Gata6-directed differentiation of NC to SMCs as an essential mechanism that specifies the aortic tree, and provide a new framework for how mutations in GATA6 lead to congenital heart disorders in humans.

Hoxa2 selectively enhances Meis binding to change a branchial arch ground state.

Hox transcription factors (TFs) are essential for vertebrate development, but how these evolutionary conserved proteins function in vivo remains unclear. Because Hox proteins have notoriously low binding specificity, they are believed to bind with cofactors, mainly homeodomain TFs Pbx and Meis, to select their specific targets. We mapped binding of Meis, Pbx, and Hoxa2 in the branchial arches, a series of segments in the developing vertebrate head. Meis occupancy is largely similar in Hox-positive and -negative arches. Hoxa2, which specifies second arch (IIBA) identity, recognizes a subset of Meis prebound sites that contain Hox motifs. Importantly, at these sites Meis binding is strongly increased. This enhanced Meis binding coincides with active enhancers, which are linked to genes highly expressed in the IIBA and regulated by Hoxa2. These findings show that Hoxa2 operates as a tissue-specific cofactor, enhancing Meis binding to specific sites that provide the IIBA with its anatomical identity.

Functional analysis of hox genes in zebrafish.

The zebrafish model organism is well suited to study the role of specific genes, such as hox genes, in embryogenesis and organ function. The ability to modulate the activity of hox genes in living zebrafish embryos represents a cornerstone of such functional analyses. In this chapter we outline the basic methodology for nucleic acid injections into 1-2-cell-stage zebrafish embryos. We also report variations in this method to allow injection of mRNA, DNA, and antisense oligonucleotides to either overexpress, knock down, or knock out specific genes in zebrafish embryos.

TALE factors poise promoters for activation by Hox proteins.

Hox proteins form complexes with TALE cofactors from the Pbx and Prep/Meis families to control transcription, but it remains unclear how Hox:TALE complexes function. Examining a Hoxb1b:TALE complex that regulates zebrafish hoxb1a transcription, we find maternally deposited TALE proteins at the hoxb1a promoter already during blastula stages. These TALE factors recruit histone-modifying enzymes to promote an active chromatin profile at the hoxb1a promoter and also recruit RNA polymerase II (RNAPII) and P-TEFb. However, in the presence of TALE factors, RNAPII remains phosphorylated on serine 5 and hoxb1a transcription is inefficient. By gastrula stages, Hoxb1b binds together with TALE factors to the hoxb1a promoter. This triggers P-TEFb-mediated transitioning of RNAPII to the serine 2-phosphorylated form and efficient hoxb1a transcription. We conclude that TALE factors access promoters during early embryogenesis to poise them for activation but that Hox proteins are required to trigger efficient transcription.

Loss of a negative feedback loop involving pea3 and cyclin d2 is required for pea3-induced migration in transformed mammary epithelial cells.

UNLABELLED: The Ets family transcription factor Pea3 (ETV4) is involved in tumorigenesis especially during the metastatic process. Pea3 is known to induce migration and invasion in mammary epithelial cell model systems. However, the molecular pathways regulated by Pea3 are still misunderstood. In the current study, using in vivo and in vitro assays, Pea3 increased the morphogenetic and tumorigenic capacity of mammary epithelial cells by modulating their cell morphology, proliferation, and migration potential. In addition, Pea3 overexpression favored an epithelial-mesenchymal transition (EMT) triggered by TGF-ß1. During investigation for molecular events downstream of Pea3, Cyclin D2 (CCND2) was identified as a new Pea3 target gene involved in the control of cellular proliferation and migration, a finding that highlights a new negative regulatory loop between Pea3 and Cyclin D2. Furthermore, Cyclin D2 expression was lost during TGF-ß1-induced EMT and Pea3-induced tumorigenesis. Finally, restored Cyclin D2 expression in Pea3-dependent mammary tumorigenic cells decreased cell migration in an opposite manner to Pea3. As such, these data demonstrate that loss of the negative feedback loop between Cyclin D2 and Pea3 contributes to Pea3-induced tumorigenesis. IMPLICATIONS: This study reveals molecular insight into how the Ets family transcription factor Pea3 favors EMT and contributes to tumorigenesis via a negative regulatory loop with Cyclin D2, a new Pea3 target gene.

Reduced tumorigenesis in mouse mammary cancer cells following inhibition of Pea3- or Erm-dependent transcription.

Pea3 and Erm are transcription factors expressed in normal developing branching organs such as the mammary gland. Deregulation of their expression is generally associated with tumorigenesis and particularly breast cancer. By using RNA interference (RNAi) to downregulate the expression of Pea3 and/or Erm in a mammary cancer cell line, we present evidence for a role of these factors in proliferation, migration and invasion capacity of cancer cells. We have used different small interfering RNAs (siRNAs) targeting pea3 and erm transcripts in transiently or stably transfected cells, and assessed the physiological behavior of these cells in in vitro assays. We also identified an in vivo alteration of tumor progression after injection of cells that overexpress pea3 and/or erm short hairpin RNAs (shRNAs) in immunodeficient mice. Using transcriptome profiling in Pea3- or Erm-targeted cells, two largely independent gene expression programs were identified on the basis of their shared phenotypic modifications. A statistically highly significant part of both sets of target genes had previously been already associated with the cellular signaling pathways of the ;proliferation, migration, invasion' class. These data provide the first evidence, by using endogenous knockdown, for pivotal and complementary roles of Pea3 and Erm transcription factors in events crucial to mammary tumorigenesis, and identify sets of downstream target genes whose expression during tumorigenesis is regulated by these transcription factors.